Methods for lowering serum cholesterol and inhibiting smooth muscle cell proliferation, restenosis, endometriosis, and uterine fibroid disease

ABSTRACT

The present invention provides novel methods of lowering serum cholesterol and inhibiting smoother muscle cell proliferation, particularly restenosis, in humans, and inhibiting uterine fibroid disease and endometriosis in women comprising administering to a human/woman in need of treatment an effective amount of a compound of formula I ##STR1## wherein R is C 1  -C 6  alkyl, C 1  -C 6  alkoxy, halo, or trifluoromethyl; 
     R 1  and R 2  each are the same or different C 1  -C 6  alkyl group; 
     n is an integer from 2 to 6; and 
     R 3  and R 4  each are independently C 1  -C 4  alkyl, or combine to form a substituent selected from the group consisting of pyrrolidino, morpholino, piperidino, piperazino, 4-(C 1  -C 6  alkyl)piperazino, and 4-phenyl-piperazino; or a pharmaceutically acceptable salt thereof.

This application is a divisional of application Ser. No. 08/419,230,filed Apr. 10, 1995, now U.S. Pat. No. 5,843,976, which is a Divisionalof Ser. No. 08/330,755, filed Oct. 23, 1994, now U.S. Pat. No.5,453,442, which is a Divisional of Ser. No. 08/198,456, filed Feb. 18,1994, now U.S. Pat. No. 5,407,955.

The present invention relates to the discovery that a group of3,4-(diphenyl)chromans are useful for lowering serum cholesterol andinhibiting smooth muscle cell proliferation, particularly, restenosis,in humans, and inhibiting endometriosis and uterine fibroid disease inwomen.

All mammalian cells require cholesterol as a structural component oftheir cell membranes and for nonsterol end products. The very property,however, that makes cholesterol useful in the cell membranes, itsinsolubility in water, also makes it potentially lethal. Whencholesterol accumulates in the wrong place, for example within the wallof an artery, it cannot be readily mobilized and its presence leads tothe development of an atherosclerotic plaque. Elevated concentrations ofserum cholesterol associated with low density lipoproteins (LDL'S) havebeen demonstrated to be a major contributing factor in the developmentand progression of atherosclerosis.

Estrogen, particularly when taken orally, lowers plasma levels of LDLand raises those of the beneficial high density lipoproteins (HDL's).Long-term estrogen therapy, however, has been implicated in a variety ofdisorders, including an increase in the risk of uterine cancer andpossibly breast cancer, causing many women to avoid this treatment.Recently suggested therapeutic regimens which seek to lessen the cancerrisk, such as administering combinations of progestin and estrogen,cause the patient to experience unacceptable bleeding. Furthermore,combining progestin with estrogen seems to blunt the serum cholesterollowering effects of estrogen. The significant undesirable effectsassociated with estrogen therapy support the need to develop alternativetherapies for hyperlipidemia/hypercholesterolemia that have thedesirable effect on serum LDL but do not cause undesirable effects.

Attempts to fill this need by the use of compounds commonly known asantiestrogens which interact with an estrogen receptor and/or bind withwhat has been termed the antiestrogen binding site (AEBS) have hadlimited success, perhaps due to the fact that these compounds generallydisplay a mixed agonist/antagonist effect and are subject to the sameadverse effects associated with estrogen therapy.

The present invention provides methods for lowering serum cholesterollevels without the associated adverse effects of estrogen therapy, andthus, provides an effective and acceptable treatment forhyperlipidemia/hypercholesterolemia.

Smooth muscle cell proliferation plays an important role in diseasessuch as atherosclerosis and restenosis. Vascular restenosis afterpercutaneous transluminal coronary angioplasty (PTCA) has been shown tobe a tissue response characterized by an early and late phase. The earlyphase, occurring hours to days after PTCA, is due to thrombosis withsome vasospasms while the late phase appears to be dominated byexcessive proliferation and migration of smooth muscle cells. In thisdisease, the increased cell motility and colonization by smooth musclecells and macrophages contribute significantly to the pathogenesis ofthe disease. The excessive proliferation and migration of vascularsmooth muscle cells may be the primary mechanism to the reocclusion ofcoronary arteries following PTCA, atherectomy, laser angioplasty, andarterial bypass graft surgery. See "Intimal Proliferation of SmoothMuscle Cells as an Explanation for Recurrent Coronary Artery Stenosisafter Percutaneous Transluminal Coronary Angioplasty," Austin et al.,Journal of the American College of Cardiology 8: 369-375 (August 1985).

Vascular restenosis remains a major long term complication followingsurgical intervention of blocked arteries by percutaneous transluminalcoronary angioplasty (PTCA), atherectomy, laser angioplasty, andarterial bypass graft surgery. In about 35% of the patients who undergoPTCA, reocclusion occurs within three to six months after the procedure.The current strategies for treating vascular restenosis includemechanical intervention by devices such as stents or pharmacologictherapies including heparin, low molecular weight heparin, coumarin,aspirin, fish oil, calcium antagonist, steroids, and prostacyclin. Thesestrategies have failed to curb the reocclusion rate and have beenineffective for the treatment and prevention of vascular restenosis. See"Prevention of Restenosis after Percutaneous Transluminal CoronaryAngioplasty: The Search for a `Magic Bullet`," Hermans et al., AmericanHeart Journal 122: 171-187 (July 1991).

In the pathogenesis of restenosis, excessive cell proliferation andmigration occurs as a result of growth factors produced by cellularconstituents in the blood and the damaged arterial vessel wall whichmediate the proliferation of smooth muscle cells in vascular restenosis.

Agents that inhibit the proliferation and/or migration of smooth musclecells are useful in the treatment and prevention of restenosis. Thepresent invention provides for the use of compounds as smooth musclecell proliferation inhibitors.

Uterine fibroid disease (uterine fibrosis) is an old and ever presentclinical problem which goes under a variety of names, including uterinehypertrophy, uterine lieomyomata, myometrial hypertrophy, fibrosisuteri, and fibrotic metritis. Essentially, uterine fibroid disease is acondition where there is an inappropriate deposition of fibroid tissueon the wall of the uterus.

This condition is a cause of dysmenorrhea and infertility in women. Theexact cause of this condition is poorly understood but evidence suggeststhat it is an inappropriate response of fibroid tissue to estrogen. Sucha condition has been produced in rabbits by daily administrations ofestrogen for 3 months. In guinea pigs, the condition has been producedby daily administration of estrogen for four months. Further, in rats,estrogen causes similar hypertrophy.

The most common treatment of uterine fibroid disease involves surgicalprocedures which are both costly and sometimes a source of complicationssuch as the formation of abdominal adhesions and infections. In somepatients, initial surgery is only a temporary treatment and the fibroidsregrow. In those cases, a hysterectomy is performed which effectivelyends the fibroids, but also the reproductive life of the patient. Also,gonadotropin releasing hormone antagonists may be administered, buttheir use is tempered by the fact they can lead to osteoporosis.

Endometriosis is a condition of severe dysmenorrhea, which isaccompanied by severe pain, bleeding into the endometrial masses orperitoneal cavity, and often leads to infertility. The cause of thesymptoms of this condition appear to be ectopic endometrial growthswhich respond inappropriately to normal hormonal control and are locatedin inappropriate tissues. Because of the inappropriate locations forendometrial growth, the tissue seems to initiate local inflammatory-likeresponses causing macrophage infiltration and a cascade of eventsleading to initiation of the painful response. The exact etiology ofthis disease is not well understood and its treatment by hormonaltherapy is diverse, poorly defined, and marked by numerous unwanted andperhaps dangerous side effects.

One of the treatments for this disease is the use of low dose estrogento suppress endometrial growth through a negative feedback effect oncentral gonadotropin release, and subsequent ovarian production ofestrogen. However, it is sometimes necessary to use continuous estrogento control the symptoms. This use of estrogen can often lead toundesirable side effects and even the risk of endometrial cancer.

Another treatment consists of continuous administration of progestinwhich induces amenorrhea and, by suppressing ovarian estrogenproduction, can cause regressions of the endometrial growths. The use ofchronic progestin therapy is often accompanied by the unpleasant centralnervous system side effects of progestin, and often leads to infertilitydue to suppression of ovarian function.

A third treatment consists of the administration of weak androgens,which are effective in controlling the endometriosis. However, theyinduce severe masculinizing effects. Several of these treatments havealso been implicated in causing a mild degree of bone loss withcontinued therapy.

Therefore, new methods of treating endometriosis are desirable.

SUMMARY OF THE INVENTION

The present invention relates to methods for lowering serum cholesteroland inhibiting smooth muscle cell proliferation and restenosiscomprising administering to a human in need of treatment an effectiveamount of a compound of formula I ##STR2## wherein

R is C₁ -C₆ alkyl, C₁ -C₆ alkoxy, halo, or trifluoromethyl;

R¹ and R² each are the same or different C₁ -C₆ alkyl group;

n is an integer from 2 to 6; and

R³ and R⁴ each are independently C₁ -C₄ alkyl, or combine to form asubstituent selected from the group consisting of pyrrolidino,morpholino, piperidino, piperazino, 4-(C₁ -C₆ alkyl)piperazino, and4-phenyl-piperazino; or a pharmaceutically acceptable salt thereof.

The present invention further relates to methods for inhibiting uterinefibroid disease and endometriosis in women comprising administering to awoman in need of treatment an effective amount of a compound of formulaI above, or a pharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns methods for lowering serum cholesterollevels, and inhibiting smooth muscle cell proliferation, restenosis,uterine fibroid disease, and endometriosis. The term "inhibit" isdefined to include its generally accepted meaning which includesprophylactically treating a subject from incurring one or more of thesedisease states, holding in check the symptoms of such a disease state,and/or treating such symptoms. Thus, the present methods include bothmedical therapeutic and/or prophylactic treatment, as appropriate.

The methods of this invention are practiced by administering to anindividual in need of treatment an effective amount of a compound offormula I ##STR3## wherein

R is C₁ -C₆ alkyl, C₁ -C₆ alkoxy, halo, or trifluoromethyl;

R¹ and R² each are the same or different C₁ -C₆ alkyl group;

n is an integer from 2 to 6; and

R³ and R⁴ each are independently C₁ -C₄ alkyl, or combine to form asubstituent selected from the group consisting of pyrrolidino,morpholino, piperidino, piperazino, 4-(C₁ -C₆ alkyl)piperazino, and4-phenyl-piperazino; or a pharmaceutically acceptable salt thereof.

The general chemical terms used in the description of a compound offormula I have their usual meanings. For example, the term "alkyl" byitself or as part of another substituent means a straight or branchedaliphatic chain having the stated number of carbon atoms such as, forexample, methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl,isohexyl, and the like. Likewise, the term "alkoxy" means an alkyl groupof the stated number of carbon atoms attached through an oxygen bridgeincluding, for example, methoxy, ethoxy, propoxy, n-propoxy, isopropoxy,and the like.

The term "halo" includes bromo, chloro, fluoro, and iodo.

Compounds of formula I are known in the art and essentially are preparedvia the methods described in U.S. Pat. Nos. 3,340,276 and 3,822,287,which are herein incorporated by reference.

U.S. Pat. No. describes, inter alia, the 3,4-diphenyl-chromans used inthe methods of the present invention. However, the process thereindisclosed prepares both the less or negligably active cis isomers aswell as the substantially more biologically active trans isomers of suchcompounds. It is preferred, however, to employ the processes disclosedin U.S. Pat. No. 3,822, 287 for preparation of the trans isomers whichare used in the methods of the present invention.

Preferred formula I compounds include those in which R is alkoxy,especially methoxy, R¹ and R² each are C₁ -C₆ alkyl, especially methyl,n is 2 or 3, especially 2, and R³ and R⁴ combine to form pyrrolidino,morpholino, and piperidino, especially pyrrolidino. A compound offormula I in which each of the especially preferred substituents is usedis known in the art as centochroman.

Although the free-base form of formula I compounds can be used in themethods of the present invention, it is preferred to prepare and use apharmaceutically acceptable salt form. Thus, the compounds used in themethods of this invention form pharmaceutically acceptable acid and baseaddition salts with a wide variety of organic and inorganic acids andbases, and include the physiologically acceptable salts which are oftenused in pharmaceutical chemistry. Such salts are also part of thisinvention. Typical inorganic acids used to form such salts includehydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric,hypophosphoric, and the like. Salts derived from organic acids, such asaliphatic mono and dicarboxylic acids, phenyl substituted alkanoicacids, hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids,aliphatic and aromatic sulfonic acids, may also be used. Suchpharmaceutically acceptable salts thus include acetate, phenylacetate,trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate,o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate,phenylbutyrate, β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate,caprate, caprylate, chloride, cinnamate, citrate, formate, fumarate,glycollate, heptanoate, hippurate, lactate, malate, maleate,hydroxymaleate, malonate, mandelate, mesylate, nicotinate,isonicotinate, nitrate, oxalate, phthalate, terephthalate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, propiolate, propionate, phenylpropionate, salicylate,sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite,bisulfite, sulfonate, benzenesulfonate, p-bromophenylsulfonate,chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate,methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,p-toluenesulfonate, xylenesulfonate, tartarate, and the like. Apreferred salt is the hydrochloride salt.

The pharmaceutically acceptable acid addition salts are typically formedby reacting a compound of formula I with an equimolar or excess amountof acid. The reactants are generally combined in a mutual solvent suchas diethyl ether or benzene. The salt normally precipitates out ofsolution within about one hour to 10 days and can be isolated byfiltration or the solvent can be stripped off by conventional means.

Bases commonly used for formation of salts include ammonium hydroxideand alkali and alkaline earth metal hydroxides, carbonates, as well asaliphatic and primary, secondary and tertiary amines, aliphaticdiamines. Bases especially useful in the preparation of addition saltsinclude ammonium hydroxide, potassium carbonate, methylamine,diethylamine, ethylene diamine and cyclohexylamine.

The pharmaceutically acceptable salts of formula I compounds generallyhave enhanced solubility characteristics compared to the compound fromwhich they are derived, and thus are often more amenable to formulationas liquids or emulsions.

Once prepared, the free base or salt form of formula I compounds can beadministered to an individual in need of treatment for the methodsherein described. The following non-limiting test examples illustratethe methods of the present invention.

Test Procedure General Preparation Procedure

In the examples illustrating the methods, a post-menopausal model wasused in which effects of different treatments upon circulating lipidswere determined.

Seventy-five day old female Sprague Dawley rats (weight range of 200 to225g) were obtained from Charles River Laboratories (Portage, Mich.).The animals were either bilaterally ovariectomized (OVX) or exposed to aSham surgical procedure at Charles River Laboratories, and then shippedafter one week. Upon arrival, they were housed in metal hanging cages ingroups of 3 or 4 per cage and had ad libitum access to food (calciumcontent approximately 0.5%) and water for one week. Room temperature wasmaintained at 22.2°±1.70° C. with a minimum relative humidity of 40%.The photoperiod in the room was 12 hours light and 12 hours dark.

Dosing Regimen Tissue Collection

After a one week acclimation period (therefore, two weeks post-OVX)daily dosing with test compound was initiated. 17α-ethynyl estradiol andthe test compound were given orally, unless otherwise stated, as asuspension in 20% cyclodextrin. Animals were dosed daily for 4 days.Following the dosing regimen, animals were weighed and anesthetized witha ketamine: Xylazine (2: 1, V:V) mixture and a blood sample wascollected by cardiac puncture. The animals were then sacrificed byasphyxiation with CO₂, the uterus was removed through a midlineincision, and a wet uterine weight was determined.

Cholesterol Analysis

Blood samples were allowed to clot at room temperature for 2 hours, andserum was obtained following centrifugation for 10 minutes at 3000 rpm.Serum cholesterol was determined using a Boehringer Mannheim Diagnosticshigh performance cholesterol assay. Briefly the cholesterol was oxidizedto cholest-4-en-3-one and hydrogen peroxide. The hydrogen peroxide wasthen reacted with phenol and 4-aminophenazone in the presence ofperoxidase to produce a p-quinonoe imine dye, which was readspectrophotemetrically at 500 nm. Cholesterol concentration was thencalculated against a standard curve. The entire assay was automatedusing a Biomek Automated Workstation.

Uterine Eosinophil Peroxidase (EPO) Assay

Uteri were kept at 4° C. until time of enzymatic analysis. The uteriwere then homogenized in 50 volumes of 50 mM Tris buffer (pH-8.0)containing 0.005% Triton X-100. Upon addition of 0.01% hydrogen peroxideand 10 mM o-phenylenediamine (final concentrations) in Tris buffer,increase in absorbance was monitored for one minute at 450 nm. Thepresence of eosonophils in the uterus is an indication of estrogenicactivity of a compound. The maximal velocity of a 15 second interval wasdetermined over the initial, linear portion of the reaction curve.

Source of Compound

17α-ethynyl estradiol was obtained from Sigma Chemical Co., St. Louis,Mo.

Influence of Formula I Compounds on Serum Cholesterol and Determinationof Agonist/Non-Agonist Activity

Data presented in Table 1 below shows comparative results amongovariectomized rats, rats treated with 17α-ethynyl estradiol (EE₂ ; anorally available form of estrogen), and rats treated with a compound ofthe present invention (centochroman). Although EE₂ caused a decrease inserum cholesterol when orally administered at 0.1 mg/Kg/day, it alsoexerted a stimulatory action on the uterus so that EE₂ uterine weightwas substantially greater than the uterine weight of ovariectomized testanimals. This uterine response to estrogen is well recognized in theart.

Not only did the compound of the present invention substantially reduceserum cholesterol compared to the ovariectomized control animals, butthe elevation of uterine weight was less than that observed with EE₂.Compared to estrogenic compounds known in the art, the benefit of serumcholesterol reduction without overtly estrogenic effects on uterineweight is quite rare and desirable.

As is expressed in the below data, estrogenicity also was assessed byevaluating the adverse response of eosinophil infiltration into theuterus. The compounds of the present invention caused a moderateincrease in the number of eosinophils observed in the stromal layer ofovariectomized rats, while EE₂ caused a substantial, expected increasein eosinophil infiltration.

The data presented in the following Table reflects the response of 5rats per treatment.

                  TABLE 1    ______________________________________                     Uterine            Serum                     Weight     Uterine Cholesterol             Dose    (% increase                                EPO     (% decrease    Compound mg/kg   vs. OVX)   (V. max)                                        vs. OVX)    ______________________________________    EE.sub.2 0.1     244.1      108.2   99.0    Centchroman             0.1     54.1       14.4    43.7             1.0     100.4      52.4    70.3             10.0    79.6       60.8    54.4    ______________________________________

In addition to the demonstrated benefits of the compounds used in themethods of the present invention, no deleterious toxicological effects(survival) were observed with any treatment.

Uterine Fibrosis Test Procedures

Test 1

Between 3 and 20 women having uterine fibrosis are administered acompound of the present invention. The amount of compound administeredis from 0.1 to 1000 mg/day, and the period of administration is 3months.

The women are observed during the period of administration, and up to 3months after discontinuance of administration, for effects on uterinefibrosis.

Test 2

The same procedure is used as in Test 1, except the period ofadministration is 6 months.

Test 3

The same procedure is used as in Test 1, except the period ofadministration is 1 year.

Test 4

A. Induction of Fibroid Tumors in Guinea Pig

Prolonged estrogen stimulation is used to induce leiomyomata in sexuallymature female guinea pigs. Animals are dosed with estradiol 3-5 timesper week by injection for 2-4 months or until tumors arise. Treatmentsconsisting of a compound of the invention or vehicle is administereddaily for 3-16 weeks and then animals are sacrificed and the uteriharvested and analyzed for tumor regression.

B. Implantation of Human Uterine Fibroid Tissue in Nude Mice

Tissue from human leiomyomas are implanted into the peritoneal cavityand or uterine myometrium of sexually mature, castrated, female, nudemice. Exogenous estrogen are supplied to induce growth of the explantedtissue. In some cases, the harvested tumor cells are cultured in vitroprior to implantation. Treatment consisting of a compound of the presentinvention or vehicle is supplied by gastric lavage on a daily basis for3-16 weeks and implants are removed and measured for growth orregression. At the time of sacrifice, the uteri is harvested to assessthe status of the organ.

Test 5

A. Tissue from Human Uterine Fibroid Tumors is Harvested and Maintained,in vitro, as Primary Nontransformed Cultures

Surgical specimens are pushed through a sterile mesh or sieve, oralternately teased apart from surrounding tissue to produce a singlecell suspension. Cells are maintained in media containing 10% serum andantibiotic. Rates of growth in the presence and absence of estrogen aredetermined. Cells are assayed for their ability to produce complementcomponent C3 and their response to growth factors and growth hormone. Invitro cultures are assessed for their proliferative response followingtreatment with progestins, GnRH, a compound of the present invention andvehicle. Levels of steroid hormone receptors are assessed weekly todetermine whether important cell characteristics are maintained invitro. Tissue from 5-25 patients are utilized.

Activity in at least one of the above tests indicates the compounds ofthe present invention are of potential in the treatment of uterinefibrosis.

Endometriosis Test Procedure

In Tests 1 and 2, effects of 14-day and 21-day administration ofcompounds of the present invention on the growth of explantedendometrial tissue can be examined.

Test 1

Twelve to thirty adult CD strain female rats are used as test animals.They are divided into three groups of equal numbers. The estrous cycleof all animals is monitored. On the day of proestrus, surgery isperformed on each female. Females in each group have the left uterinehorn removed, sectioned into small squares, and the squares are looselysutured at various sites adjacent to the mesenteric blood flow. Inaddition, females in Group 2 have the ovaries removed.

On the day following surgery, animals in Groups 1 and 2 receiveintraperitoneal injections of water for 14 days whereas animals in Group3 receive intraperitoneal injections of 1.0 mg of a compound of thepresent invention per kilogram of body weight for the same duration.Following 14 days of treatment, each female is sacrificed and theendometrial explants, adrenals, remaining uterus, and ovaries, whereapplicable, are removed and prepared for histological examination. Theovaries and adrenals are weighed.

Test 2

Twelve to thirty adult CD strain female rats are used as test animals.They are divided into two equal groups. The estrous cycle of all animalsis monitored. On the day of proestrus, surgery is performed on eachfemale. Females in each group have the left uterine horn removed,sectioned into small squares, and the squares are loosely sutured atvarious sites adjacent to the mesenteric blood flow.

Approximately 50 days following surgery, animals assigned to Group 1receive intraperitoneal injections of water for 21 days whereas animalsin Group 2 receive intraperitoneal injections of 1.0 mg of a compound ofthe present invention per kilogram of body weight for the same duration.Following 21 days of treatment, each female is sacrificed and theendometrial explants and adrenals are removed and weighed. The explantsare measured as an indication of growth. Estrous cycles are monitored.

Test 3

A. Surgical Induction of Endometriosis

Autographs of endometrial tissue are used to induce endometriosis inrats and/or rabbits. Female animals at reproductive maturity undergobilateral oophorectomy, and estrogen is supplied exogenously thusproviding a specific and constant level of hormone. Autologousendometrial tissue is implanted in the peritoneum of 5-150 animals andestrogen supplied to induce growth of the explanted tissue. Treatmentconsisting of a compound of the present invention is supplied by gastriclavage on a daily basis for 3-16 weeks, and implants are removed andmeasured for growth or regression. At the time of sacrifice, the intacthorn of the uterus is harvested to assess status of endometrium.

B. Implantation of Human Endometrial Tissue in Nude Mice

Tissue from human endometrial lesions is implanted into the peritoneumof sexually mature, castrated, female, nude mice. Exogenous estrogen issupplied to induce growth of the explanted tissue. In some cases, theharvested endometrial cells are cultured in vitro prior to implantation.Treatment consisting of a compound of the present invention supplied bygastric lavage on a daily basis for 3-16 weeks, and implants are removedand measured for growth or regression. At the time of sacrifice, theuteri is harvested to assess the status of the intact endometrium.

Test 4

A. Tissue from Human Endometrial Lesions is Harvested and Maintained invitro as Primary Nontransformed Cultures

Surgical specimens are pushed through a sterile mesh or sieve, oralternately teased apart from surrounding tissue to produce a singlecell suspension. Cells are maintained in media containing 10% serum andantibiotic. Rates of growth in the presence and absence of estrogen aredetermined. Cells are assayed for their ability to produce complementcomponent C3 and their response to growth factors and growth hormone. Invitro cultures are assessed for their proliferative response followingtreatment with progestins, GnRH, a compound of the invention, andvehicle. Levels of steroid hormone receptors are assessed weekly todetermine whether important cell characteristics are maintained invitro. Tissue from 5-25 patients is utilized.

Activity in any of the above assays indicates that the compounds of thepresent invention are useful in the treatment of endometriosis.

Inhibition of Vascular Smooth Cell Proliferation/Restenosis TestProcedure

Compounds of the present invention have capacity to inhibit vascularsmooth cell proliferation. This can be demonstrated by using culturedsmooth cells derived from rabbit aorta, proliferation being determinedby the measurement of DNA synthesis. Cells are obtained by explantmethod as described in Ross, J. of Cell Bio. 50: 172 (1971). Cells areplated in 96 well microtiter plates for five days. The cultures becomeconfluent and growth arrested. The cells are then transferred toDulbecco's Modified Eagle's Medium (DMEM) containing 0.5-2% plateletpoor plasma, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg mlstreptomycin, 1 mC/ml ³ H-thymidine, 20 ng/ml platelet-derived growthfactor, and varying concentrations of the present compounds. Stocksolution of the compounds is prepared in dimethyl sulphoxide and thendiluted to appropriate concentration (0.01-30 mM) in the above assaymedium. Cells are then incubated at 37° C. for 24 hours under 5% CO₂/95% air. At the end of 24 hours, the cells are fixed in methanol. ³ Hthymidine incorporation in DNA is then determined by scintillationcounting as described in Bonin, et al., Exp. Cell Res. 181: 475-482(1989).

Inhibition of smooth muscle cell proliferation by the compounds of thepresent invention are further demonstrated by determining their effectson exponentially growing cells. Smooth muscle cells from rabbit aortaeare seeded in 12 well tissue culture plates in DMEM containing 10% fetalbovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/mlstreptomycin. After 24 hours, the cells are attached and the medium isreplaced with DMEM containing 10% serum, 2 mM L-glutamine, 100 U/mlpenicillin, 100 mg/ml streptomycin, and desired concentrations of thecompounds. Cells are allowed to grow for four days. Cells are treatedwith trypsin and the number of cells in each culture is determined bycounting using a ZM-Coulter counter.

Activity in the above tests indicates that the compounds of the presentinvention are of potential in the treatment of smooth muscle cellproliferation, particularly restenosis.

For the majority of the methods of the present invention, compounds ofFormula I are administered continuously, from 1 to 3 times daily.However, cyclical therapy may especially be useful in the treatment ofendometriosis or may be used acutely during painful attacks of thedisease. In the case of restenosis, therapy may be limited to short (1-6months) intervals following medical procedures such as angioplasty.

As used herein, the term "effective amount" means an amount of compoundof the methods of the present invention which is capable of loweringserum cholesterol and inhibiting the symptoms of the variouspathological conditions herein described. The specific dose of acompound administered according to this invention will, of course, bedetermined by the particular circumstances surrounding the caseincluding, for example, the compound administered, the route ofadministration, the state of being of the patient, and the pathologicalcondition being treated. A typical daily dose will contain a nontoxicdosage level of from about 0.5 mg to about 600 mg/day of a compound ofthe present invention. Preferred daily doses generally will be fromabout 15 mg to about 600 mg/day.

The compounds of this invention can be administered by a variety ofroutes including oral, rectal, transdermal, subucutaneus, intravenous,intramuscular, and intranasal. These compounds preferably are formulatedprior to administration, the selection of which will be decided by theattending physician. Typically, a formula I compound, or apharmaceutically acceptable salt thereof, is combined with apharmaceutically acceptable carrier, diluent or excipient to form apharmaceutical formulation.

The total active ingredients in such formulations comprises from 0.1% to99.9% by weight of the formulation. By "pharmaceutically acceptable" itis meant the carrier, diluent, excipients, and/or salt must becompatible with the other ingredients of the formulation, and notdeleterious to the recipient thereof.

Pharmaceutical formulations containing a compound of formula I can beprepared by procedures known in the art using well known and readilyavailable ingredients. For example, the compounds of formula I can beformulated with common excipients, diluents, or carriers, and formedinto tablets, capsules, suspensions, powders, and the like. Examples ofexcipients, diluents, and carriers that are suitable for suchformulations include the following: fillers and extenders such asstarch, sugars, mannitol, and silicic derivatives; binding agents suchas carboxymethyl cellulose and other cellulose derivatives, alginates,gelatin, and polyvinylpyrrolidone; moisturizing agents such as glycerol;disintegrating agents such as calcium carbonate and sodium bicarbonate;agents for retarding dissolution such as paraffin; resorptionaccelerators such as quaternary ammonium compounds; surface activeagents such as cetyl alcohol, glycerol monostearate; adsorptive carrierssuch as kaolin and bentonite; and lubricants such as talc, calcium andmagnesium stearate, and solid polyethyl glycols.

The compounds also can be formulated as elixirs or solutions forconvenient oral administration or as solutions appropriate forparenteral administration, for example, by intramuscular, subcutaneousor intravenous routes.

Additionally, the compounds are well suited to formulation as sustainedrelease dosage forms and the like. The formulations can be soconstituted that they release the active ingredient only or preferablyin a particular physiological location, possibly over a period of time.The coatings, envelopes, and protective matrices may be made, forexample, from polymeric substances or waxes.

Compounds of formula I generally will be administered in a convenientformulation. The following formulation examples only are illustrativeand are not intended to limit the scope of the present invention.

Formulations

In the formulations which follow, "active ingredient" means a compoundof formula I, or a salt thereof.

Formulation 1

Gelatin Capsules

Hard gelatin capsules are prepared using the following:

    ______________________________________    Ingredient        Quantity (mg/capsule)    ______________________________________    Active ingredient 0.1-1000    Starch, NF        0-650    Starch flowable powder                      0-650    Silicone fluid 350 centistokes                      0-15    ______________________________________

The formulation above may be changed in compliance with the reasonablevariations provided.

A tablet formulation is prepared using the ingredients below:

Formulation 2

Tablets

    ______________________________________    Ingredient       Quantity (mg/tablet)    ______________________________________    Active ingredient                      2.5-1000    Cellulose, microcrystalline                     200-650    Silicon dioxide, fumed                      10-650    Stearate acid     5-15    ______________________________________

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 2.5-1000 mg of active ingredientare made up as follows:

Formulation 3

Tablets

    ______________________________________    Ingredient         Quantity (mg/tablet)    ______________________________________    Active ingredient   25-1000    Starch             45    Cellulose, microcrystalline                       35    Polyvinylpyrrolidone                       4    (as 10% solution in water)    Sodium carboxymethyl cellulose                       4.5    Magnesium stearate 0.5    Talc               1    ______________________________________

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at ₅₀°-60 ° C. and passed through a No. 18 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate, and talc, previouslypassed through a No. 60 U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yieldtablets.

Suspensions each containing 0.1-1000 mg of medicament per 5 ml dose aremade as follows:

Formulation 4

Suspensions

    ______________________________________    Ingredient          Quantity (mg/5 ml)    ______________________________________    Active ingredient   0.1-1000   mg    Sodium carboxymethyl cellulose                        50         mg    Syrup               1.25       mg    Benzoic acid solution                        0.10       mL    Flavor              q.v.    Color               q.v.    Purified water to   5          mL    ______________________________________

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl cellulose and syrup to form a smooth paste. Thebenzoic acid solution, flavor, and color are diluted with some of thewater and added, with stirring. Sufficient water is then added toproduce the required volume.

An aerosol solution is prepared containing the following ingredients:

Formulation 5

Aerosol

    ______________________________________    Ingredient           Quantity (% by weight)    ______________________________________    Active ingredient    0.25    Ethanol              25.75    Propellant 22 (Chlorodifluoromethane)                         70.00    ______________________________________

The active ingredient is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to 30° C., and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remaining propellant. The valve units arethen fitted to the container.

Suppositories are prepared as follows:

Formulation 6

Suppositories

    ______________________________________    Ingredient     Quantity (mg/suppository)    ______________________________________    Active ingredient                   250    Saturated fatty acid                   2,000    glycerides    ______________________________________

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimal necessary heat. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool. Anintravenous formulation is prepared as follows:

Formulation 7

Intravenous Solution

    ______________________________________    Ingredient            Quantity    ______________________________________    Active ingredient     50     mg    Isotonic saline       1,000  mL    ______________________________________

The solution of the above ingredients is intravenously administered to apatient at a rate of about 1 mL per minute.

We claim:
 1. A method of lowering serum cholesterol which comprisesadministering to a human in need of treatment an effective amount of acompound of formula I: ##STR4## wherein R is C₁ -C₆ alkyl, C₁ -C₆alkoxy, halo, or trifluoromethyl;R¹ and R² each are the same ordifferent C₁ -C₆ alkyl group; n is an integer from 2 to 6; and R³ and R⁴each are independently C₁ -C₄ alkyl, or combine to form a substituentselected from the group consisting of pyrrolidino, morpholino,piperidino, piperazino, 4-(C₁ -C₆ alkyl)piperazino, and4-phenyl-piperazino; or a pharmaceutically acceptable salt thereof.
 2. Amethod according to claim 1 wherein R is methoxy; R₁ and R² each aremethyl; and n is 2; or a pharmaceutically acceptable salt thereof.
 3. Amethod according to claim 1 wherein R³ and R⁴ each are ethyl, or apharmaceutically acceptable salt thereof.
 4. A method according to claim1 wherein R³ and R⁴ combine to form a pyrrolidino group, or apharmaceutically acceptable salt thereof.
 5. A method according to claim1 wherein wherein R³ and R⁴ combine to form a piperidino group, or apharmaceutically acceptable salt thereof.
 6. A method according to claim1 wherein R³ and R⁴ combine to form a morpholino group, or apharmaceutically acceptable salt thereof.
 7. A method according to claim1 wherein said salt thereof is the hydrochloride salt.
 8. A method oflowering serum cholesterol which comprises administering to a human inneed of treatment an effective amount of centchroman.